Chapter I

Introduction
1.1 Background
Aphanomyces invadans is a eukaryotic pathogen and the causative agent of
Epizootic Ulcerative Syndrome (EUS) in fish and is responsible for mortalities of up
to 100% in aquaculture. A. invadans was first discovered in Japan in 1971, and since
then it has been found in Australia, North America, Southern African countries and
Asia. Methods for the correct identification of A. invadans are well established now
and involve PCR-based detection and microscopy. However, the pathogenesis of A.
invadans is poorly understood. Environmental stress (mainly temperature) and the
associated immunocompromised fish seem to induce infections of A. invadans and
outbreaks of EUS. Understanding the process of infection in more depth is
fundamental for the discovery of novel effective treatments to combat the disease. In
this review, we discuss morphological characteristics of A. Invadans and its
pathogenicity as well as various approaches of treatment.
Disease problems constitute the largest single cause of economic loss in
aquaculture. Over the past several years, the development and application of
molecular diagnostic techniques has initiated a revolution in the diagnosis and
monitoring of infectious diseases. PCR-based systems to detect the etiologic agents of
disease directly from clinical samples, without the need for culture, have been useful
in rapid detection of uncultivable or fastidious microorganisms and allows for
identification and better characterization of the pathogen.
1.2 Analysis Problem
1. What is the etiology of Aphanomyces invadans?
2. What kind of disease that caused by Aphanomyces invadans?
3. What kind identification method that can identify Aphanomyces invadans?
4. How to prevent and medicate disease caused by Aphanomyces invadans?
1.3 Analysis Objective
1. To know the etiology of Aphanomyces invadans.
2. To know kind of disease that caused by Aphanomyces invadans.
3. To know kind identification method that can identify Aphanomyces invadans.
4. To know to prevent and medicate disease caused by Aphanomyces invadans.

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 Chapter II

Discussion

2.1 General Description of Aphanomyces invadans Infection

Infection with A. invadans is a seasonal epizootic condition of great
importance in wild and farmed freshwater and estuarine fish. It has a complex
infectious aetiology and is clinically characterised by the presence of invasive A.
invadans infection and necrotising ulcerative lesions, typically leading to a
granulomatous response. Infection with A. invadansis most commonly known as
epizootic ulcerative syndrome (EUS). It is also known as red spot disease (RSD),
mycotic granulomatosis (MG) and ulcerative mycosis (UM). In 2005, scientists
proposed that the disease should be named epizootic granulomatous aphanomycosis
however, the term EUS continues to be used by most scientists. The disease is caused
by the oomycete fungus A. invadans.
Infection with Aphanomyces invadans has spread widely since the first
outbreak in 1971 in Japan and to date only one genotype has been recorded. Parasites
and rhabdoviruses have also been associated with particular outbreaks, and secondary
Gram-negative bacteria invariably infect lesions caused by A. invadans. The genus
Aphanomyces is a member of a group of organisms commonly known as the water
moulds.
Although long regarded as a fungus because of its characteristic filamentous
growth, this group, the Oomycetida, is not a member of the Eumycota, but is
classified with diatoms and brown algae in a group called the Stramenopiles or
Chromista.
2.1.1 Etiology of Aphanomyces invadans
Aphanomyces is a genus of water moulds. As of 2003 there were about 45
described species. Many of these water moulds are known as economically important
pathogens of species of plants and animals, including fish, crustaceans, and
agricultural crop plants. Aphanomyces water moulds are "one of the most important
yield-limiting factors in production of legumes and sugarbeet."
Some of these water moulds are host-specific, such as A. iridis, which only
infects irises. Others can infest several hosts, such as A. euteiches, which grows on

2
 several legumes. The disease that manifests in plants infected with these water moulds
is sometimes known simply as Aphanomyces root rot or common root rot.
Aphanomyces invadans (Saprolegniales, Oomycetes) has an aseptate fungal-
like mycelia structure. This oomycete has two typical zoospore forms. The primary
zoospore consists of round cells that develop inside the sporangium. The primary
zoospore is released to the tip of the sporangium where it forms a spore cluster. It
quickly transforms into the secondary zoospore, which is reniform with laterally
biflagellate cells and can swim freely in the water. The secondary zoospore remains
motile for a period that depends on the environmental conditions and presence of the
fish host or substratum. Typically, the zoospore encysts and germinates to produce
new hyphae, although further tertiary generations of zoospores may be released from
cysts (polyplanetism).
2.1.2 Life Cycle of Aphanomyces invadans
A. invadans passes through the typical life stages of an oomycete without the
sexual stage which is often rare or absent in animal-pathogenic oomycetes (Dick et al.,
1999). In contrast, the asexual stage is distinctive for the genus Aphanomyces, which is
characterized by the formation of biflagellate zoospores from clusters of primary cysts
at hyphal tips. Aphanomyces spp. propagate asexually by the formation of
zoosporangia comprising 30 to 50 primary zoospores which are released through lateral
evacuation tube into the environment. Interestingly, isolates from the Philippines can
have up to 2 lateral evacuation tubes per sporangium and isolates from Thailand
have normally 4 tubes per sporangium.
After release from sporangia, primary zoospores immediately encyst at the
apical tip and form achlyoid clusters. From these clusters secondary zoospore are
released which are the main free-swimming stage. Later, secondary zoospores
transform into a germling by forming a germ tube, which eventually develops into
mycelium. However, encysted zoospores of parasitic Aphanomyces species are capable
of releasing a new zoospore generation instead of germinatin. This process is called
repeated zoospore emergence (RZE) or polyplanetism.
2.1.3 Susceptible Host

Aphanomyces invadans causes disease and mortality in farmed and wild fish,
worldwide. Around 94 species of fish have been confirmed by histological diagnosis to
be naturally affected by A. invadans. Cases of natural infection with A. invadans in

3
 species other than those listed should be referred immediately to the appropriate OIE
Reference Laboratory, whether or not clinical signs are associated with the findings.
Some fish, such as common carp (Cyprinus capio), Nile tilapia (Oreochromis niloticus)
and milk fish (Chanos chanos), have been considered to be naturally resistant to
infection with A. invadans.
2.1.4 Transmission
Infection with A. invadans is transmitted horizontally. The A. invadans
zoospores can be horizontally transmitted from one fish to another through the water
supply. It is believed that only the secondary zoospores or free-swimming stage
zoospores are capable of attaching to the damaged skin of fish and germinating into
hyphae. If the secondary zoospores cannot find the susceptible species or encounter
unfavourable conditions, they can encyst in the pond environment. The cysts may
wait for conditions that favour their transformation into tertiary generations of
zoospores that are also in the free-swimming stage. The encystingproperty of A.
invadans may play an importance role in the cycle of outbreaks in endemic areas.

2.2 Epizootic Ulcerative Syndrome

2.2.1 Clinical Symptoms
Under normal conditions skin defence mechanism of fish are sufficient to
prevent an infection with A. invadans. However, a suppression of the immune system
can result in an outbreak of EUS. Various environmental determinants such as
colonisation of the fish by other pathogens (viruses, bacteria or ectoparasites), a low
pH or low oxygen concentrations in the water can affect primary defence mechanisms
of fish. Under appropriate conditions, some A. invadans isolates are capable of acting
as a primary pathogen.
Infections of fish by A. invadans are initiated by attachment of motile
zoospores. They are attracted to their host, attach to the damaged skin and form
germination tubes that penetrate the skin. Hyphae invade deep into lower tissues
resulting in extensive ulceration and destruction of the tissue. The typical signs of
ulceration can be used as a presumptive diagnosis for EUS on affected fish. The
occurrence of skin lesions varies depending on the fish species as well as
manifestation and ranges from crucial areas of intense inflammation and hyperaemia
of the skin to deep ulcerations with uncovering of the lower muscle tissue. Early

4
 lesions are characterized by haemorrhagic bullae with small foci of ulceration and are
often observed on the lateral surface. Concomitantly, scales are often protruding.
In contrast, lesions in bluegills are characterised by proliferative tan areas on
the skin which are associated with scale loss. Mildly infected fish show only minor
inflammation without external lesions but petechial haemorrhagic spots on body,
mouth as well as anal fin and exophtalmia. In severe cases, swollen haemorrhagic
areas, massive inflammation and large deep ulcerations in association with necrosis of
the myotome can be observed. Ulcers appear as white areas on the skin of the fish
with reddish centres that turn into complete red areas and later result in external
haemorrhages and distended abdomens. As the disease progresses, eyes are
protruding, the body becomes putrefactive and in some cases also the head is eroded,
which results in the death of fish due to the severity of the disease.
2.2.2 Pathology

An infection with A. invadans is histologically characterised by severe necrosis

of the muscle tissue around the invading hyphae which later develop into enclosed
granulomas. Whereas non-invasive aseptic hyphae with a smaller diameter are
frequently observed at the surface of the site of infection. Migrating hyphae are
observed along fascial layers and between myofibrils where they cause myconecrosis.
The severe lesions attract large numbers of inflammatory cells which results in
oedema and hyperplasia of gill lamella, but only marginal changes ofinternal organs.
Most of the haematological data revealed that fish with EUS show a significant
increase of white blood cells (especially neutrophils) due to a local inflammatory
response, while the number of red blood cells, the haemoglobin concentration and the
haematocrit level are reduced because of the blood loss caused by the haemorrhagic
lesions. The combination of an increase of neutrophils and a reduction of erythrocytes
(including haematocrit and haemoglobin content) shows that A. invadans-infected fish
suffer from anaemic condition.

2.2.3 Identification of Aphanomyces invadans

 PCR-based
A very sensitive and specific technique is ITS sequencing (Internal Transcribes
Spacer) which is widely used for the molecular identification and differentiation of
oomycetes from species to genus level. The three main lineages of A. invadans can

5
 be also distinguished by ITS sequencing. In addition, restriction fragment length
polymorphisms (RFLPs) and sequences of ribosomal DNA (rDNA) were used to
distinguish A. invadans from other species including saprolegniaceae. Recently,
also the mitochondrial gene cytochrome c oxidase subunit I (COI) was used for the
identification of Aphanomyces species. The mitochondrial genome was sequenced
recently and found to be 49,061 basepairs long and has a large inverted repeat
region of about 12 kb.
Comparison of ITS and COI sequences of 23 oomycete genera including
Aphanomyces sp. revealed, that both are suitable for the identification of
oomycetes but in some cases COI was more discriminative than ITS at the species
level. However, COI sequences are not conserved throughout all Aphanomyces
species.
 Microscopy-based
Mycelia-like structures and zoospores of A. invadans can be detected by in situ
hybridisation or immunofluorescent staining on histological sections of infected
fish.
 Immunological Method
Various immunological methods were established to reliably identify A. invadans
with a very high sensitivity (7 mg/ml, dilutions up to 10-11) such as FTA
(Monoclonal antibody-based flow-through immunoassay) or immunoblots.
2.2.4 Treatment
 External Chemical Treatment
Protozoan parasites are often quite abundant and sometimes pose a potential threat
to the health of fishes. Ichthyophthirius multifiliis, an external protozoan parasite
(white spot), is quite difficult to treat in its encysted form. Hence, mixed treatment
with malachite green and formalin is often recommended. Conversely, the
monogenetic trematodes, notably the Gyrodactylus and the crustacean parasites;
for example, the Lernaea and the Argulus, can be treated with organophosphates,
mainly because they do not seem to be susceptible to formalin.
 Systemic Treatment via Diet
Concomitant to the above, it is often considered important to achieve a correct rate
of dosage in the quantity of medicated feed offered to the fish. In fact, a balance
has preferably to be reached between toxic and therapeutic levels in the

6
 bloodstream. Nevertheless, the feeding rate is said to depend on a number of
factors, including the activity of the drug, water temperature, and the time of year.
Usually, healthy fish are reluctant to accept medicated diets. There could also be
variations in the acceptance of medicated diets by the diseased fish. It could
sometimes be difficult to correctly ascertain, how much diet the fish could actually
consume. Moreover, the availability of drugs could prove difficult in certain
countries, and drugs might be prescribed by registered veterinary practitioners
only. In fact, such regulations and restrictions are considered significant because
they help in reducing the risk of development of resistant strains of pathogenic
bacteria. Likewise, physical incorporation of drugs into the diet could also present
practical difficulties. Concomitantly, there could be strict limitations of marketing
of fishes following a course of medicated feeding. There are of course no
legislative requirements, but still the producers are expected to be under a moral
obligation not to harvest such fish for at least four weeks after treatment, in order
to allow the antibiotic to be cleared from the tissues of the fish. Otherwise, such
fish could be responsible for developing antibiotic resistance in the bacterial flora
of the person consuming them.
2.2.5 Prevention
A. Better water-quality management, as in aquaculture practices
B. Application of unslaked lime in unaffected ponds at 200 kg/ha once a month for
three consecutive months, depending on the pH of the water and soil
C. Restrictions on transferring the use of nets and other potentially transmitting agents
from affected to unaffected water bodies
D. Fish from infected waterways, especially those with lesions of EUS, are not to be
relocated to other water bodies
E. Prophylactic measure, salt, potassium permanganate, bleaching powder, and
malachite green may be recommended as alternative or additional prophylactic
measures
2.2.6 Control
a. Control of EUS in natural waters (e.g., rivers and wetlands) is very difficult, as
indicated.
b. Fish farmers whose farmed fish have been affected by EUS may be
encouraged to culture non-EUS susceptible fishes, or to avoid farming
susceptible species during the EUS outbreak period.

7
c. A strict ban on the movement of fish from the affected water bodies to other
unaffected water bodies is recommended.
d. Diseased fishes are not to move from one fish farm to another.
e. Properly dried, salted, and iced fish may not be potential carriers of EUS. As
such, trade with such fishes and their products could be allowed to continue.
f. Further, a number of simple biosecurity measures coulminimize or prevent the
spread of EUS.

8
 Chapter III

Conclusion

Infection with A. invadans is a seasonal epizootic condition of great importance in

wild and farmed freshwater and estuarine fish. It has a complex infectious aetiology and is
clinically characterised by the presence of invasive A. invadans infection and necrotising
ulcerative lesions, typically leading to a granulomatous response. Infection with A. invadansis
most commonly known as epizootic ulcerative syndrome (EUS). It is also known as red spot
disease (RSD), mycotic granulomatosis (MG) and ulcerative mycosis (UM).

There are some kind of identification of Aphanomyces invadans. First is PCR-based,

microscopy-based and the last is immunological method. Treatment of Aphanomyces
invadans are divided into two, external chemical treatment and systemic treatment via diet.
Prevention of this disease are better water-quality management, as in aquaculture practices,
application of unslaked lime in unaffected ponds at 200 kg/ha once a month for three
consecutive months, depending on the ph of the water and soil, restrictions on transferring the
use of nets and other potentially transmitting agents from affected to unaffected water bodies,
fish from infected waterways, especially those with lesions of eus, are not to be relocated to
other water bodies, prophylactic measure, salt, potassium permanganate, bleaching powder,
and malachite green may be recommended as alternative or additional prophylactic measures.

9
 BIBLIOGRAPHY

Sibanda, Sitokozile et all. (2018). Emergence of infection with Aphanomyces invadans in

fish in some main aquatic ecosystems in Zimbabwe: A threat to national fisheries
production. Zimbabwe, University of Zimbabwe. Zimbabwe.

Iberahim, Nurul Aqilah., Trusch, Franziska., Van West, Pieter. (2018) Aphanomyces
invadans, the causal agent of Epizootic Ulcerative Syndrome, is a global threat to
wild and farmed fish. In: Fungal Biological Reviews. Page 1-12. British
Mycological Society. British.

Afzali, Fatameh et all. (2015) Detecting Aphanomyces Invadans in Pure Cultures and EUS-
infected Fish Lesions by Applying PCR. In: Malaysian Journal of Medical and
Biological Research. Page 137-146. Asian Business Consortium. Malaysia

OIE. (2018). Infection With Aphanomyces Invadans (Epizootic Ulcerative Syndrome) In:
Manual of Diagnostic Tests for Aquatic Animals. 2.3.2 Page 2-13.

Huchzermeyer CF, Huchzermeyer KDA, Christison KW, et al. First-record of epizootic

ulcerative syndrome from the Upper Congo catchment: An outbreak in the
Bangweulu swamps, Zambia. J Fish Dis. 2017;00:1–8.

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